Abstract. Medullary thyroid carcinoma (MTC), a neuroen-docrine tumor originating from thyroid parafollicular cells, has been demonstrated to be associated with mutations in
RET, HRAS, KRAS and NRAS. However, the role of other
genes involved in the oncogenesis of neural crest tumors remains to be fully investigated in MTC. The current study aimed to investigate the presence of somatic mutations in
BRAF, CDKN2A and PI3KCA in MTC, and to investigate
the correlation with disease progression. DNA was isolated from paraffin-embedded tumors and blood samples from patients with MTC, and the hotspot somatic mutations were sequenced. A total of 2 novel HRAS mutations, p.Asp33Asn and p.His94Tyr, and polymorphisms within the 3' untranslated region (UTR) of CDKN2A (rs11515 and rs3088440) were identified, however, no mutations were observed in other genes. It was suggested that somatic point mutations in BRAF,
CDKN2A and PI3KCA do not participate in the oncogenesis
of MTC. Further studies are required in order to clarify the contribution of the polymorphisms identified in the 3'UTR of
CDKN2A in MTC. Introduction
Medullary thyroid carcinoma (MTC), a neuroendocrine tumor originating from thyroid parafollicular cells, accounts for ~4% of thyroid cancer cases (1). The majority are sporadic cases, however, 20-25% occur as a hereditary syndrome termed multiple endocrine neoplasia type 2 (MEN 2A and MEN 2B)
and as familial MTC, both of which are associated with germ-line mutations in the RET oncogene (2).
Mutations in the RET oncogene have previously been identi-fied in the tumor tissue of up to 64% of sporadic MTC cases (3). In addition, RAS gene mutations are observed in 10% of
RET-negative cases and are associated with a subset of tumors
with less aggressive behavior (4). While certain studies identified that ~90% of sporadic MTCs exhibited mutually exclusive muta-tions in RET, HRAS and KRAS (4-8), Moura et al (3) reported the presence of the RAS mutation in one case with RET-positive sporadic MTC and Rapa et al (9) identified no RAS mutations in 49 examined cases. Nevertheless, the clinical phenotype of sporadic and inherited MTCs is heterogeneous even in the pres-ence of the same mutation; however the molecular mechanisms underlying the pathology remain to be fully elucidated.
In addition, it remains unclear whether there is a modula-tory role in MTC tumor progression for additional genes such as BRAF, CDKN2A and PI3KCA. These genes participate in the tumorigenesis of several types of human malignancies such as tumors derived from neural crest cells, including melanoma, pheochromocytoma and paraganglioma (10-12).
BRAF, like RET and RAS, is involved in the
mitogen-activated protein kinase pathway and has a well-established role in the pathogenesis of malignancies such as melanoma and papillary thyroid cancer (13). Nevertheless, the contribution in the tumorigenesis of MTC remains contro-versial. A previous study reported a high prevalence of the p.Val600Glu BRAF mutation in sporadic MTC cases (14); however, subsequent studies did not confirm this observa-tion (3,9,15,16).
An additional tumor suppressor gene, CDKN2A/p16INK4A, is
involved in the G1/S transition in the cell cycle. Mutations and
deletions have been identified in melanoma, and polymorphisms in its 3' untranslated region (UTR) have been associated with earlier progression from primary to metastatic disease (17). By contrast, polymorphisms in another tumor suppressor gene,
CDKN1B, which is in the same CDKN family, are associated
with improved outcomes (18).
Additionally, PI3KCA is a gene that serves an important role in signaling pathways and cell growth, and contributes to tumorigenesis in several types of human malignancy (19,20).
Analysis of somatic mutations in BRAF, CDKN2A/p16 and
PI3KCA in patients with medullary thyroid carcinoma
FABRÍCIO P. NASCIMENTO1, MIRIAN G. CARDOSO1,2, SUSAN C. LINDSEY1, ILDA S. KUNII1, FLÁVIA O.F. VALENTE1, MARINA M.L. KIZYS1, ROSANA DELCELO3,
CLÉBER P. CAMACHO1, RUI M.B. MACIEL1 and MAGNUS R. DIAS-DA-SILVA1,2
1Laboratory of Molecular and Translational Endocrinology, Department of Medicine;
Departments of 2Biochemistry and 3Pathology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil
Received December 31, 2014; Accepted October 14, 2015 DOI: 10.3892/mmr.2015.4731
Correspondence to: Professor Magnus R. Dias-da-Silva, Laboratory of Molecular and Translational Endocrinology, Department of Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo, 669 Rua Pedro de Toledo, São Paulo 04039-032, Brazil E-mail: mrdsilva@unifesp.br
Key words: medullary thyroid cancer, somatic mutation, RET,
However, the role of this gene in the tumorigenesis of MTC remains to be fully understood.
Therefore, the current study aimed to verify the prevalence of somatic mutations in BRAF, CDKN2A and PI3KCA, which have already been described in other neural crest-derived tumors, and to determine the possible supporting role of these genes in the tumorigenesis of MTC.
Patients and methods
Patients and tissue samples. From 128 patients with MTC
assessed at the Multiple Endocrine Neoplasia outpatient clinic at the Universidade Federal de Sao Paulo (Sao Paulo, Brazil) between February 2007 and June 2013, formalin‑fixed paraffin‑embedded (FFPE) tumor tissues were selected from 31 patients on the basis of the availability of tumor tissues, with no other selection criteria. DNA extraction was subse-quently performed, using an in-house method as previously described (21). Subsequent to DNA extraction, 20 samples (from 13 males and 7 females; mean age, 40.55±16.74 years) provided the appropriate quantity and quality of DNA. The study was approved by the Ethics and Research Committee of the Universidade Federal de Sao Paulo (protocol number 1945/10), and all patients provided informed consent. Additionally, 1,092 genotypes of variant frequencies (single nucleotide polymorphisms; SNPs) were obtained from the 1000 Genomes database (http://www.1000genomes.org/) as a population genetics control.
DNA extraction and genotyping. DNA from peripheral
blood and somatic DNA from 10-µm sections of FFPE MTC tissues was extracted using an in-house method as previously described (21). Polymerase chain reaction (PCR) was performed to amplify DNA corresponding to hotspot exons 2, 3 and 4 of HRAS; 2, 3 and 4 of KRAS; 2 and 3 of
NRAS; 15 of BRAF; 9 and 20 of PI3KCA; and exons 2, 3 and
the 3'UTR of the CDKN2A gene. The sequences of the primers are listed in Table I. The reactions were performed using 10 pM of each specific primer, 2.5 µl PCR buffer, 200 µM dNTP, 1.5 µM MgCl2 and 0.2 units Taq DNA
poly-merase (Invitrogen; Thermo Fisher Scientific, Waltham, MA, USA) in a 25-µl total reaction volume. The cycling condi-tions were as follows: 5 min at 95˚C, 38 cycles of 45 sec at 95˚C, 45 sec for annealing and 1 min at 72˚C, and a final elongation for 10 min at 72˚C. The PCR products were puri-fied using the Illustra GFX PCR DNA and Gel Purification kit (GE Healthcare Life Sciences, Chalfont, UK) and were subject to sequencing using the Sanger method, with the Big Dye™ Terminator Cycle Sequencing Ready Reaction kit and the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific). Gel electrophoresis of the PCR products was performed to analyze product quality and yield using a 1.8% agarose gel and a DNA ladder.
In silico analysis of HRAS mutations and CDKN2A polymor-phisms. Mutational analysis of HRAS was performed by the
use of Project HOPE to obtain structural information from the analysis of PDB‑file 1CTQ (22). The in silico analysis for the
CDKN2A polymorphisms was performed using the Functional
Single Nucleotide Polymorphism database (http://compbio. cs.queensu.ca/F-SNP/) as previously described (23). This database provides information regarding potential deleterious effects of SNPs with respect to splicing, transcription, transla-tion and post-translatransla-tion based on SNP functransla-tional significance (FS). The FS score for neutral SNPs is 0.1764, whereas the FS score for disease-associated SNPs is in the range of 0.5-1.
Statistical analysis. The allele and genotype frequencies were
compared between patients with MTC and the 1000 Genomes database controls using a χ2 test. The clinicopathological features
of patients carrying each of the polymorphisms rs11515 and rs3088440 were compared with those of patients without such Table I. Primers used in the present study.
Gene Forward primer Reverse primer
BRAF exon 15 5'-AACTCAGCAGCATCTCAGGG-3' 5'-CTTCATAATGCTTGCTCTGATAG-3'
CDKN2A exon 1 5'-ACCCTGGCTCTGACCATTC-3' 5'-CAGGTCACGGGCAGAC-3'
CDKN2A exon 2 5'-GACCTCAGGTTTCTAACGCC-3' 5'-CATATATCTACGTTAAAAGGCAGGAC-3'
PI3KCA exon 9 5'-TGGCAGTCAAACCTTCTCTC-3' 5'-GAGAAAGTATCTACCTAAATCCACAGA-3'
PI3KCA exon 20 5'-AAATGTTTTGGTGTTCTTAATTTATTC-3' 5'-GCAGCCAGAACTCTTTATTTTG-3'
C-kit exon 9 5'-GCCAGGGCTTTTGTTTTCTT-3' 5'-AGCCTAAACATCCCCTTAAATTG-3'
C-kit exon 11 5'-AACCATTTATTTGTTCTCTCTCCA-3' 5'-CCACTGGAGTTCCTTAAAGTCA-3'
C-kit exon 17 5'-TGGTTTTCTTTTCTCCTCCAAC-3' 5'-GGACTGTCAAGCAGAGAATGG-3'
HRAS exon 2 5'-GGCAGGAGACCCTGTAGGAG-3' 5'-AGCTGCTGGCACCTGGAC-3'
HRAS exon 3 5'-GTCCCTGAGCCCTGTCCTC-3' 5'-CAGCCTCACGGGGTTCAC-3'
HRAS exon 4 5'-CTCTCGCTTTCCACCTCTCA-3' 5'-GGGTGGAGAGCTGCCTCA-3'
KRAS exon 2 5'-TTAACCTTATGTGTGACATGTTCTAA-3' 5'-GGTCCTGCACCAGTAATATGC-3'
KRAS exon 3 5'-AGACTGTGTTTCTCCCTTCTCA-3' 5'-TGGCATTAGCAAAGACTCAAA-3'
KRAS exon 4 5'-GATATTTGTGTTACTAATGACTGTGCT-3' 5'-TTATGATTTTGCAGAAAACAGATC-3'
NRAS exon 2 5'-TCGCCAATTAACCCTGATTAC-3' 5'-TCCGACAAGTGAGAGACAGG-3'
polymorphisms using the χ2 test or the Student's unpaired t-test
as appropriate. P<0.05 was considered to indicate a statistically significant difference, and the Hardy-Weinberg equilibrium was evaluated. Statistical analyses were performed using SPSS, version 22.0 (IBM SPSS, Armonk, NY, USA) and GraphPad Prism, version 3.0 (GraphPad Software, Inc., La Jolla, CA, USA). Results
Screening of the RET, HRAS, KRAS and NRAS genes.
Mutational screening of the RET gene was performed on all 20 patients. A total of 10 cases were identified to be familial tumors as confirmed by the presence of a germline mutation. In total, 30% of the sporadic cases (3/10) presented with a RET somatic mutation. The clinicopathological features and molecular analysis, including tumor staging based on the American Joint Committee in Cancer staging system (24), are summarized in Table II.
To investigate exclusive causative mutations in cases of sporadic MTC other than RET mutations, HRAS, KRAS and NRAS were screened for somatic mutations in the hotspots. The majority of these patients had been previously analyzed for RET germline mutations as part of our routine evalua-tion, and for RET somatic mutations in a previous study (25) Two novel HRAS mutations, p.Asp33Asn and p.His94Tyr, were detected in RET-negative MTC tumors. Mutational analysis using Project HOPE suggests that the p.His94Tyr mutation is deleterious, and that the p.Asp33Asn mutation is likely to be
damaging (Fig. 1). No differences in the clinical presentation or histological observations were noted between patients with MTC that had a mutation in the RAS gene (Table II).
No somatic mutations were identified in exon 15 of BRAF or in exons 9 and 20 of PI3KCA. Patient 9 was not analyzed for somatic mutations in PI3KCA due to an insufficient number of tumor samples.
Despite not having identified somatic mutations in
CDKN2A hotspots, two polymorphisms in the 3'UTR
regula-tory region, 500 C→G (rs11515) and 540 C→T (rs3088440), were identified in the patients observed. The heterozygotic pattern of the two SNPs was observed in the same propor-tion, 7/20 MTC (35%). The genotype distribution was identified to be in the Hardy-Weinberg equilibrium and was not identi-fied to exhibit linkage disequilibrium. To investigate whether the observed polymorphisms were limited to a somatic event, they were further analyzed in the peripheral blood, which confirmed germline inheritance. The in silico analysis demonstrated that the CDKN2A polymorphisms rs11515 and rs3088440 are located in the transcriptional regulatory region and that the nucleotide alterations may affect the binding of transcription factors.
In seven cases, it was possible to detect the presence of these polymorphisms in the secondary tumors in the lymph nodes (tumor metastases), however no differences between the genotypes of the primary and secondary tumors were observed, indicating that there was no additional somatic event in CDKN2A involved in the metastatic process. This analysis Table II. Summary of patient clinicopathological features and molecular analysis.
Age at Germline RET Somatic Somatic H-, K-, Somatic
Patient Gender diagnosis (y) pTNMa allele RET allele NRAS allele CDKN2A
1 M 28 T2N1bMx WT WT HRAS_p.Asp33Asn rs11515 2 F 25 T3N1bMx WT p.Met918Thr - WT 3 M 38 T1N1aMx WT WT NA rs11515/rs3088440 4 M 56 T3N1bMx WT WT WT WT 5 F 49 T2NxMx WT p.Gln681Stop - WT 6 M 69 T2N0Mx WT WT HRAS_p.Gln61Arg rs3088440 7 M 27 T4N1Mx WT WT WT WT 8 M 51 T3N1bMx WT p.Met918Thr - rs11515 9 F 56 T1N1bMx WT WT HRAS_p.Asp33Asn WT 10 M 41 T4N1bMx WT WT HRAS_p.His94Tyr WT 11 M 27 T1N1aMx p.Cys634Arg - - rs11515/rs3088440 12 F 21 T1N1aMx p.Gly533Cys - - rs11515 13 M 61 T1N1aMx p.Gly533Cys - - WT 14 F 22 T2N0Mx p.Cys634Arg - - rs11515/rs3088440 15 M 43 T2N0Mx p.Cys634Arg - - rs11515 16 M 72 T1N0Mx p.Cys634Arg - - WT 17 M 45 T1N1aMx p.Cys634Arg - - rs3088440 18 F 31 T1NxMx p.Cys634Arg - - rs3088440 19 F 15 T1N1aMx p.Cys634Arg - - rs3088440 20 M 40 T1N0Mx p.Gly533Cys - - WT
aTNM (Tumor, Node, Metastasis)/American Joint Committee on Cancer staging system. M, male; F, female; y, years; NA, not available; WT,
was additionally performed for BRAF and PI3KCA in meta-static tissues.
No associations between the polymorphisms and the clini-copathological features observed were identified (Table III). In addition, the frequency of the SNPs was compared with a population genetics control, and there was no significant difference between the two populations (Table IV).
Discussion
The adjuvant role of additional genes in the tumorigenesis of MTC was investigated in the current study through analysis of tumor tissues from 20 patients. Screening in hotspot regions of BRAF, CDKN2A and PI3KCA did not identify any somatic mutations in the coding region. In addition, the results of the current study were not in agreement with the BRAF muta-tion frequency of 68.2% observed by Goutas et al (14). This suggests that BRAF does not serve an important role in the
tumorigenesis of MTC. The observations of the current study concerning MTC are consistent with a previous study that demonstrated that somatic mutations in genes other than RET and RAS are very rare or even absent (5). Notably, the present study identified two novel HRAS mutations.
Additionally, two common polymorphisms in the 3'-UTR non-coding region of the gene CDKN2A were identi-fied, rs11515 and rs3088440 (26). It is known that protein synthesis can be modulated by regulatory elements located in the 5'-UTR and 3'-UTR regions. The 3'-UTR, the site of the polymorphisms identified in the current study, serves an important role in translation and mRNA stability. Alterations in this region may be associated with the onset or progression of disease (27).
These polymorphisms have been investigated in various tumor types including urinary bladder neoplasm (28), esophageal adenocarcinoma (29) and cervical cancer (30) as presented in Table V. The two identified polymorphisms have
Figure 1. Mutational analysis of the HRAS somatic mutations p.Asp33Asn and p.His94Tyr. Electropherogram of tumor tissues (A) 1 and (B) 10; (C and D) sequence alignment of human HRAS protein residues in which the position of the conserved amino acids are indicated (arrows); multiple sequence alignment was generated with Clustal Omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/), *indicates that the residues in the column were identical in all sequences
in the alignment; schematic structures of the (E) original and (F) mutant amino acids in the two HRAS mutations; (G and H) structure of the HRAS proteins in ribbon-presentation; gray, protein; magenta, side chain of the mutation (p.Asp33Asn and p.His94Tyr).
A B
C D
E F
Table III. Correlation between CDKN2A SNPs and clinicopathological features in the patient cohort.
rs11515 (n=20) rs3088440 (n=20)
Clinicopathological ---
---feature CC (n=13) CG (n=7) P-value CC (n=13) CT (n=7) P-value
Gender 0.526 0.474 Male (n=13) 8/13 (61.5%) 5/7 (71.4%) 9/13 (69.2%) 4/7 (57.1%) Female (n=7) 5/13 (38.5%) 2/7 (28.6%) 4/13 (30.7%) 3/7 (42.9%) Age at diagnosis 0.088a 0.272a Mean ± SD (y) 45.41±17.49 31.53±11.36 44.062±17.49 31.53±11.36 Tumor type 0.500 0.175 Sporadic (n=10) 7/13 (53.8%) 3/7 (42.9%) 8/13 (61.5%) 2/7 (28.5%) Familial (n=10) 6/13 (46.1%) 4/7 (57.1%) 5/13 (38.5%) 5/7 (62.5%) T category 0.464 0.291 T1 7/13 (53.8%) 2/7(28.5%) 5/13 (38.5%) 4/7 (57.1%) T2 2/13 (15.3%) 3/7 (42.9%) 3/13 (23.1%) 2/7 (28.5%) T3 2/13 (15.3%) 2/7 (28.5%) 3/13 (23.1%) 1/7 (14.4%) T4 2/13 (15.3%) 0/7 (0%) 2/13 (15.3%) 0/7 (0%) Tumor size 0.421a 0.689a Mean ± SD (cm) 1.954±1.11 2.34±1.03 2.315±1.22 1.671±0.59 <2 8/13 (61.5%) 2/7(28.6%) 0.378 7/13 (53.8%) 4/7 (57.1%) 0.339 ≥2 5/13 (38.5%) 5/7 (71.4%) 6/13 (46.1%) 3/7 (42.9%)
Lymph node metastases 0.742 0.742
N0 4/13 (30.8%) 5/7 (71.4%) 3/13 (23.07%) 3/7 (42.9%)
N1 9/13 (69.2%) 2/7 (28.5%) 10/13 (76.9%) 4/7 (57.1%)
AJCC stage 0.742 0.742
I and II 4/13 (30.7%) 2/7 (28.5%) 4/13 (30.7%) 2/7 (28.5%)
III and IV 9/13 (69.2%) 5/7 (71.4%) 9/13 (69.2%) 5/7 (71.4%)
P-values were obtained using the χ2 test; acontinuous variables analyzed with Student's t-test. SNPs, single nucleotide polymorphisms; SD,
standard deviation; y, years; AJCC, American Joint Committee on Cancer.
Table IV. Comparative analysis of the frequency of the non-coding CDKN2A germ line single nucleotide polymorphisms in patients with MTC and the control.
A, rs11515
Genotype frequency Allele frequency
---
---Population CC CG GG C (32) G (8) P-value
MTC 0.60 0.40 - 0.80 0.20 0.25
1,000 genomesa 0.79 0.19 0.02 0.88 0.12
B, rs3088440
Genotype frequency Allele frequency
---
---Population CC CT TT C (31) T (9) P-value
MTC 0.55 0.45 - 0.78 0.22 0.65
1,000 genomesa 0.73 0.24 0.03 0.85 0.15
aSequences obtained from the 1000 Genomes database used as a population control. MTC, medullary thyroid carcinoma. The numbers in
been previously associated with an earlier progression from primary to metastatic disease in the case of melanoma (17), and rs3088440 was associated with the mechanism of tumor invasion in bladder cancer (28). Controversially, this poly-morphism has been previously associated with a sub-group with reduced vertical growth of melanoma and a favorable outcome (31). However, additional studies have not identified a clinical correlation with tumor behavior (30,32,33).
Using in silico analysis, the current study identified that the polymorphisms rs11515 and rs3088440 are located within a transcriptional regulatory region, and the alteration of nucleotides can affect the binding of potential transcrip-tional factors. For example, the presence of the C allele in rs3088440 favors the binding of the transcription factor c-Myb, which potentially results in the transcriptional repression of the CDKN2A gene, compromising its normal function in cell cycle control (42). However, no association was identified between this polymorphism and the clinico-pathological parameters investigated in the cohort studied (Table III).
In conclusion, it is suggested that BRAF, CDKN2A and
PI3KCA, listed as potential adjuvants in the tumorigenesis
of MTC, do not participate through somatic mutations as modulators of oncogenesis. To the best of our knowledge, the current study is the first to investigate these two CDKN2A
polymorphisms in the pathophysiology of MTC. Therefore,
CDKN2A and its regulatory regions and the additional genes
involved in tumorigenesis warrant further investigation in MTC.
Acknowledgements
The authors would like to thank the team of the Laboratory of Molecular and Translational Endocrinology, particu-larly Ms. Teresa Kasamatsu, Mr. Gilberto Furuzawa, Dr João Roberto Martins, Dr Ji Hoon Yang, Dr Fausto Germano Neto and Mr. Fernando Soares. The authors would additionally like to acknowledge Mr. Gilmar Miranda from Siratec Ltd. for graphic art design. The current study was supported grants from the Sao Paulo State Research Foundation (grant nos. 2012/11036-3, 2012/02465-8, 2012/01628-0, 2009/50575-4, 2012/00079-3 and 2011/20747-8).
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